Cr(vi) permanently binds to the lipid bilayer in an inverted hexagonal phase throughout the reduction process.

Cr(vi) is a harmful, carcinogenic agent with a high permeability rate throughout the lipid membranes. In an intracellular environment and during interactions with cellular membranes, it undergoes an instant reduction to lower oxidation states throughout radical states, recognized as the most dangerous factor for cells. The cellular membrane is the most visible cellular organelle in the interior and exterior of a cell. In this study, liposomes and non-lamellar inverted hexagonal phase lipid structures based on phosphoethanolamine (PE) were used as model cellular bilayers because of their simple composition, preparation procedure, and the many other properties of natural systems. The lipid membranes were subjected to 0.075 mM Cr(vi) for 15 min, after which the Cr content was removed via dialysis. This way, the remaining Cr content could be studied qualitatively and quantitatively. Using the combined XRF/XAS/EPR approach, we revealed that some Cr content (Cr(iii) and Cr(vi)) was still present in the samples even after long-term dialysis at a temperature significantly above the phase transition for the chosen liposome. The amount of bound Cr increased with increasing PE and -C[double bond, length as m-dash]C- bond content in lipid mixtures. Internal membrane order decreased in less fluid membranes, while in more liquified ones, internal order was only slightly changed after subjecting them to the Cr(vi) agent. The results suggest that the inverted hexagonal phase of lipid structures is much more sensitive to oxidation than the lamellar lipid phase, which can play an important role in the strong cytotoxicity of Cr(vi).

Release systems based on self-assembling RADA16-I hydrogels with a signal sequence which improves wound healing processes.

Self-assembling peptides can be used for the regeneration of severely damaged skin. They can act as scaffolds for skin cells and as a reservoir of active compounds, to accelerate scarless wound healing. To overcome repeated administration of peptides which accelerate healing, we report development of three new peptide biomaterials based on the RADA16-I hydrogel functionalized with a sequence (AAPV) cleaved by human neutrophil elastase and short biologically active peptide motifs, namely GHK, KGHK and RDKVYR. The peptide hybrids were investigated for their structural aspects using circular dichroism, thioflavin T assay, transmission electron microscopy, and atomic force microscopy, as well as their rheological properties and stability in different fluids such as water or plasma, and their susceptibility to digestion by enzymes present in the wound environment. In addition, the morphology of the RADA-peptide hydrogels was examined with a unique technique called scanning electron cryomicroscopy. These experiments enabled us to verify if the designed peptides increased the bioactivity of the gel without disturbing its gelling processes. We demonstrate that the physicochemical properties of the designed hybrids were similar to those of the original RADA16-I. The materials behaved as expected, leaving the active motif free when treated with elastase. XTT and LDH tests on fibroblasts and keratinocytes were performed to assess the cytotoxicity of the RADA16-I hybrids, while the viability of cells treated with RADA16-I hybrids was evaluated in a model of human dermal fibroblasts. The hybrid peptides revealed no cytotoxicity; the cells grew and proliferated better than after treatment with RADA16-I alone. Improved wound healing following topical delivery of RADA-GHK and RADA-KGHK was demonstrated using a model of dorsal skin injury in mice and histological analyses. The presented results indicate further research is warranted into the engineered peptides as scaffolds for wound healing and tissue engineering.

The Process of Binding and Releasing of Genetic Material from Lipoplexes Based on Trimeric Surfactants and Phospholipids.

Nonviral vectors for gene therapy such as lipoplexes are characterized by low toxicity, high biocompatibility, and good transfection efficiency. Specifically, lipoplexes based on polymeric surfactants and phospholipids have great potential as gene carriers due to the increased ability to bind genetic material (multiplied positive electric charge) while lowering undesirable effects (the presence of lipids makes the system more like natural membranes). This study aimed to test the ability to bind and release genetic material by lipoplexes based on trimeric surfactants and lipid formulations of different compositions and to characterize formed complexes by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). The cytotoxicity of studied lipoplexes was tested on HeLa cells by the MTT cell viability assay and the dye exclusion test (trypan blue). The presence of lipids in the system lowered the surfactant concentration required for complexation (higher efficiency) and reduced the cytotoxicity of lipoplexes. Surfactant/lipids/DNA complexes were more stable than surfactant/DNA complexes. Surfactant molecules induced the genetic material condensation, but the presence of lipids significantly intensified this process. Systems based on trimeric surfactants and lipid formulations, particularly TRI_N and TRI_IMI systems, could be used as delivery carrier, and have proven to be highly effective, nontoxic, and universal for DNA of various lengths.

Comprehensive and comparative studies on nanocytotoxicity of glyceryl monooleate- and phytantriol-based lipid liquid crystalline nanoparticles.

BACKGROUND: Lipid liquid crystalline nanoparticles (LLCNPs) emerge as a suitable system for drug and contrast agent delivery. In this regard due to their unique properties, they offer a solubility of a variety of active pharmaceutics with different polarities increasing their stability and the possibility of controlled delivery. Nevertheless, the most crucial aspect underlying the application of LLCNPs for drug or contrast agent delivery is the unequivocal assessment of their biocompatibility, including cytotoxicity, genotoxicity, and related aspects. Although studies regarding the cytotoxicity of LLCNPs prepared from various lipids and surfactants were conducted, the actual mechanism and its impact on the cells (both cancer and normal) are not entirely comprehended. Therefore, in this study, LLCNPs colloidal formulations were prepared from two most popular structure-forming lipids, i.e., glyceryl monooleate (GMO) and phytantriol (PHT) with different lipid content of 2 and 20 w/w%, and the surfactant Pluronic F-127 using the top-down approach for further comparison of their properties. Prepared formulations were subjected to physicochemical characterization and followed with in-depth biological characterization, which included cyto- and genotoxicity towards cervical cancer cells (HeLa) and human fibroblast cells (MSU 1.1), the evaluation of cytoskeleton integrity, intracellular reactive oxygen species (ROS) generation upon treatment with prepared LLCNPs and finally the identification of internalization pathways. RESULTS: Results denote the higher cytotoxicity of PHT-based nanoparticles on both cell lines on monolayers as well as cellular spheroids, what is in accordance with evaluation of ROS activity level and cytoskeleton integrity. Detected level of ROS in cells upon the treatment with LLCNPs indicates their insignificant contribution to the cellular redox balance for most concentrations, however distinct for GMO- and PHT-based LLCNPs. The disintegration of cytoskeleton after administration of LLCNPs implies the relation between LLCNPs and F-actin filaments. Additionally, the expression of four genes involved in DNA damage and important metabolic processes was analyzed, indicating concentration-dependent differences between PHT- and GMO-based LLCNPs. CONCLUSIONS: Overall, GMO-based LLCNPs emerge as potentially more viable candidates for drug delivery systems as their impact on cells is not as deleterious as PHT-based as well as they were efficiently internalized by cell monolayers and 3D spheroids.

Functionalized Peptide Fibrils as a Scaffold for Active Substances in Wound Healing.

Technological developments in the field of biologically active peptide applications in medicine have increased the need for new methods for peptide delivery. The disadvantage of peptides as drugs is their low biological stability. Recently, great attention has been paid to self-assembling peptides that can form fibrils. Such a formulation makes bioactive peptides more resistant to enzymatic degradation and druggable. Peptide fibrils can be carriers for peptides with interesting biological activities. These features open up prospects for using the peptide fibrils as long-acting drugs and are a valid alternative to conventional peptidic therapies. In our study, we designed new peptide scaffolds that are a hybrid of three interconnected amino acid sequences and are: pro-regenerative, cleavable by neutrophilic elastase, and fibril-forming. We intended to obtain peptides that are stable in the wound environment and that, when applied, would release a biologically active sequence. Our studies showed that the designed hybrid peptides show a high tendency toward regular fibril formation and are able to release the pro-regenerative sequence. Cytotoxicity studies showed that all the designed peptides were safe, did not cause cytotoxic effects and revealed a pro-regenerative potential in human fibroblast and keratinocyte cell lines. In vivo experiments in a dorsal skin injury model in mice indicated that two tested peptides moderately promote tissue repair in their free form. Our research proves that peptide fibrils can be a druggable form and a scaffold for active peptides.